Bottom Line:
Preliminary results indicated that phenoloxidase is primarily responsible for this virucidal effect.Addition of the enzyme superoxide dismutase to plasma did not affect virucidal activity; however addition of catalase had an inhibitory effect.Inhibitors of nitric oxide synthase activity did not affect virucidal activity.

ABSTRACTHeliothis virescens larval plasma contains high levels of an antiviral activity against the budded form of the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) in vitro. Preliminary results indicated that phenoloxidase is primarily responsible for this virucidal effect. However it is known that other enzymes that generate antimicrobial reactive oxygen intermediates and reactive nitrogen intermediates are present in hemolymph that could contribute to the observed virucidal activity. To elucidate the contributions of phenoloxidase and other candidate activities to plasma innate immune response against baculovirus infection specific metabolic inhibitors were used. In vitro the general inhibitors of melanization (N-acetyl cysteine, ascorbate and glutathione), and specific inhibitors of phenoloxidase (phenylthiourea and Kojic acid), completely blocked virucidal activity up to the level seen in controls. Addition of the enzyme superoxide dismutase to plasma did not affect virucidal activity; however addition of catalase had an inhibitory effect. Inhibitors of nitric oxide synthase activity did not affect virucidal activity. Our results confirm that phenoloxidase is the predominate activity in larval plasma accounting for inactivation of HzSNPV in vitro, and that phenoloxidase-dependent H(2)O(2) production may contribute to this virucidal activity.

Mentions:
Addition of serine proteinase inhibitors, that prevent the activation of HvPPO to HvPO, and of the inhibitor phenylthiourea, that chelates copper from the active site of the enzyme, was previously demonstrated to inhibit plasma virucidal activity in larval H. virescens plasma (Popham et al. 2004). To further elucidate the enzymatic nature of the virucidal activity, additional specific inhibitors of HvPO activity were sought. Kojic acid (5-hydroxy-2-hydroxymethyl-γ-pyrone) is a specific competitive inhibitor of insect phenoloxidases (Dowd 1988; Chen et al. 1991a; Chen et al. 1991b; Li and Kubo 2004). Preincubation of diluted early 5th instar H. virescens plasma with 1 mM Kojic acid completely abolished virucidal activity (n = 4, p < 0.013, Student-Newman-Keuls multiple comparison test; Fig. 2). Additionally, the copper chelator diethyldithiocarbamic acid when fed to larvae has been reported to suppress resistance to infection with the baculovirus AcMNPV (Washburn et al. 1996). This inhibitor proved to be too toxic to HzAM-1 cells and thus could not used in further experiments (data not shown).

Bottom Line:
Preliminary results indicated that phenoloxidase is primarily responsible for this virucidal effect.Addition of the enzyme superoxide dismutase to plasma did not affect virucidal activity; however addition of catalase had an inhibitory effect.Inhibitors of nitric oxide synthase activity did not affect virucidal activity.

ABSTRACTHeliothis virescens larval plasma contains high levels of an antiviral activity against the budded form of the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) in vitro. Preliminary results indicated that phenoloxidase is primarily responsible for this virucidal effect. However it is known that other enzymes that generate antimicrobial reactive oxygen intermediates and reactive nitrogen intermediates are present in hemolymph that could contribute to the observed virucidal activity. To elucidate the contributions of phenoloxidase and other candidate activities to plasma innate immune response against baculovirus infection specific metabolic inhibitors were used. In vitro the general inhibitors of melanization (N-acetyl cysteine, ascorbate and glutathione), and specific inhibitors of phenoloxidase (phenylthiourea and Kojic acid), completely blocked virucidal activity up to the level seen in controls. Addition of the enzyme superoxide dismutase to plasma did not affect virucidal activity; however addition of catalase had an inhibitory effect. Inhibitors of nitric oxide synthase activity did not affect virucidal activity. Our results confirm that phenoloxidase is the predominate activity in larval plasma accounting for inactivation of HzSNPV in vitro, and that phenoloxidase-dependent H(2)O(2) production may contribute to this virucidal activity.